CN106256835A - High-glycosylation human growth hormone's fusion protein and preparation method thereof and purposes - Google Patents

High-glycosylation human growth hormone's fusion protein and preparation method thereof and purposes Download PDF

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CN106256835A
CN106256835A CN201610694877.4A CN201610694877A CN106256835A CN 106256835 A CN106256835 A CN 106256835A CN 201610694877 A CN201610694877 A CN 201610694877A CN 106256835 A CN106256835 A CN 106256835A
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fusion protein
human
ctp
hgh
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李强
陈思
郑云程
马心鲁
李子瑞
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Anyuan Pharmaceutical Technology (shanghai) Co Ltd
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Priority to CN201710202564.7A priority patent/CN107286248B/en
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    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

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Abstract

The invention discloses a kind of high-glycosylation human growth hormone's fusion protein.Fusion protein described in human growth hormone's fusion protein of the present invention contains human growth hormone (hGH), flexible peptide linker (L), human chorion gonadotrophic hormone beta subunit carboxyl terminal rigidity peptide (CTP) and human normal immunoglobulin's Fc fragment successively from N end to C end.The invention also discloses a kind of efficient method preparing this kind of fusion protein.The fusion protein that the present invention builds has more excellent internal drug effect than restructuring hGH, the circulating half-life in vivo of prolongation, and administration frequency is substantially reduced and bioavailability improves；Its production process is simpler, efficiently simultaneously.

Description

High-glycosylation human growth hormone's fusion protein and preparation method thereof and purposes
Technical field
The present invention relates to fusion protein field, more particularly it relates to an high-glycosylation human growth hormone is merged Albumen and its production and use.
Background technology
Human growth hormone (human growth hormone, hGH) is secreted by anterior pituitary oxyphil cell Planting proteohormone, be made up of 191 aminoacid, its physiological function is chiefly to facilitate substance metabolism and growth promoter.HGH passes through Somatomedin or insulin like growth factor mediation, in cartilaginous tissue, increase bone length, thus reach to promote human body Linear growth.Recent years, the more function of hGH was found, including promoting skeletal muscle and Myocyte growth, promotion albumen The effects such as matter synthesis, regulation function of immune system and enhancing immune defence ability.The larger scale clinical of hGH is applied also from initial The prevention of the children short stature of growth hormone deficiency and treatment, be extended to the necks such as burn, acute pancreatitis, old people's slow down aging Territory, its clinical indication is also in constantly extension.
Child and adult, in the case of growth hormone deficiency, can be treated by external source supplementation with growth hormones. The internal removing half-life of natural hGH is about 0.3 hour, the internal removing half-life of current business-like recombinant human somatropin It is about 2-3 hour, liver, kidney removing will soon being passed through after injection, so needing subcutaneous administrations every day, carrying to patient Come a lot of painful.Additionally, long term injections recombinant human somatropin produces antibody in a few patients body and affects the treatment.Therefore, A kind of technology is needed to extend the activity that can also keep higher while hGH circulating half-life in vivo.Build recombinant long-acting, height The human growth hormone of activity, the immunogenicity reduced and higher bioavailability becomes the primary mesh of drug development of future generation Mark.
For stable protein, prevent the scavenging action of enzymolysis and kidney, such as Polyethylene Glycol (PEG) can be used to modify (Sada et al., J.Ferment Bioeng 71:137-139,1991), glycosylation modified transformation (U.S. Patent number US 7, 217,689) and with other protein the method for (WO 93/15199) is merged to promote body absorption, to prevent from being degraded and kidney Remove.The albumen that PEG modifies is prevented from hydrolysis, will not cause serious side effects, but PEG belongs to non-with target protein coupling Specific covalent combines, and this random coupling interaction between target protein and its receptor that hinders in various degree causes Its activity in vivo reduces.Increase glycosylation and can increase the molecular weight of target protein, filter critical line especially for being in kidney Protein (～30KD), glycosylation can reduce the protein sensitivity to protease hydrolysis.Korean Patent Publication No KR10-2013-0029713 discloses by introducing at least one sudden change addition N-glycosyl on α-1 antitrypsin, attempts increasing The method adding Half-life in vivo.The glycosylation modified of protein has two classes, including O-sugar chain and N-sugar chain.But, sugar chain attached And adding to cause physiologically active protein matter to inactivate, and can additionally adhere to the selection face in the site of the physiologically active protein matter of sugar chain The narrowest, therefore introduce the glycosylation engineering of extra glycosylation site to protein and be not also widely used.
The another kind of method improving albumen pharmacokinetics and internal stability is to be connected by the gene of physiologically active protein On the expressing gene of an albumen with high stability, by gene recombination technology to produce fusion protein.Fc merges egg Be in vain utilize the technology such as genetic engineering certain is had biologic activity functional protein molecule (soluble ligand, receptor or its He needs to extend the bioactive substance of half-life) with Fc segment composition and the novel recombinant protein that produces.Such fusion protein Not only remain the biologic activity of functional protein molecule, moreover it is possible to extend the half-life of fusion protein and improve its stability, Fc The interchain disulfide bond of fragment is conducive to fusion molecule to form dimer, thus strengthens ligand binding capacity and improve biological activity； The introducing of Fc fragment also advantageously improves fusion molecule expression in mammalian cell.Therefore manufacture and contain and people The fusion protein that the Fc region of IgG albumen is connected, it will help extend the circulating half-life of medicine and/or increase the biological alive of it Property.Patent CN102875683 discloses the Fc fusion protein of a kind of long-acting recombinant human growth hormone, becomes at hGH and human IgG Fc Body adds one section of flexible peptide linker to reduce space steric effect, it is contemplated that target is to make its extended serum half lives, biological Activity increases, thus improves pharmacokinetics and drug effect.But, owing to the avtive spot of hGH is mainly at C end, C-terminal merges Other albumen leverage activity and the function of hGH, CN102875683 patent shows the hGH-L-vFc's of dimerization Molar specific activity is 1.2nM, and it is the most not ideal enough that its activity compares restructuring hGH.Studying discovery according to the present invention, simple prolongation is soft Property peptide linker, increase Fc Yu hGH C end space length can not solve problem, need other method solve fusion part pair A difficult problem for hGH activity influence.It is contemplated that find new fusion method, improve further fusion protein serum half-life and Activity in vivo.
CTP is the small peptide of one section of β from human chorionic gonadotropin (hCG)-subunit carboxyl terminal.Four kinds and reproduction Relevant peptide hormone, follicle stimulating hormone (FSH), lutropin (LH), thyrotropin (TSH) and chorionic gonadotrophin Hormone (hCG) contains identical α-subunit and the most special β-subunit.Compared with other three kinds of hormones, hCG Half-life in vivo Being obviously prolonged, this is mainly derived from (the Fares FA et al.Proc Natl of distinctive carboxyl terminal peptide (CTP) on its β-subunit Acad Sci USA.89:4304–4308,1992).CTP contains 37 amino acid residues, and it has 4 O-glycosylation sites, Terminal is sialic acid residues.CTP can increase the level of glycosylation of protein, improves the activity of target protein, the most electronegative, The most sialylated CTP can resist the kidney scavenging action to it, thus extends the albumen half-life in vivo.The U.S. is special Profit 13,195,931 discloses a kind of method by connecting chorionic-gonadotropin hormone carboxyl terminal peptide (CTP) on hGH, makes Growth hormone circulating half-life only extends about 6 times, but and the size of not mentioned several hGH/CTP chimeric protein external activity； The display of internal drug effect is merged the chimeric protein of 1 or 2 CTP molecule and is relatively recombinated the weak effect of hGH, only CTP-hGH-CTP-CTP This chimeric protein drug effect being fitted together to 3 CTP molecules is slightly above recombinated hGH.The present inventor creatively will have multiple O- The CTP polypeptide in glycosyl site, as a part for connection peptides, is used for connecting hGH and Fc fragment rather than sending out as merging part Wave effect, the native glycosylation sites having because of it, the half-life of fusion protein can not only be made to extend further, bioavailability Improve, be also greatly reduced the fusion part Fc steric effect to hGH so that it is maintain higher biologic activity simultaneously.
Comprehensive above content, has had many protein fusion methods to be constructed to develop long acting protein class medicine, But the most still there is no activity and half-life the most gratifying long-acting hGH preparation.Therefore, this area is long in the urgent need to exploitation Effect, high activity, purification step are simple, be easy to the long-acting hGH of industrialized production.
Summary of the invention
The present invention is to solve the restructuring problem that hGH serum half-life is short and biological activity is poor, design and be prepared for one The long-acting hGH fusion protein of high-glycosylation.The present inventor, through in-depth study for a long time, devises the peptide linker of uniqueness first That reduces between fusion molecule is sterically hindered, the C end of hGH be connected the fusion protein formed with Fc, middle with flexible peptide and just Property peptide connect.Unexpectedly, the external activity of the hGH-L-vFc that this fusion protein was developed than former the present inventor improves about 1 times (seeing, China Patent No. CN102875683).Additionally, also achieve unforeseeable technique effect, the most described fusion egg White bioavailability is also greatly improved.
In a first aspect of the present invention, it is provided that a kind of restructuring hGH fusion protein, described fusion protein depends on to C end from N end Secondary containing human growth hormone (hGH), flexible peptide linker (L), human chorion gonadotrophic hormone beta-subunit carboxyl terminal rigidity peptide And human normal immunoglobulin's Fc fragment (CTP)；Wherein, Fc fragment preferred human IgG Fc variant (being expressed as vFc).
Wherein, the preferred non-immunogenic of described flexible peptide linker, and between hGH and Fc, produce enough distances, Steric effect each other is made to be down to minimum.It is preferred that use the flexible peptide containing following 2 or multiple Amino acid profile to connect Head: Gly (G), Ser (S), Ala (A) and Thr (T).Preferably, described flexible peptide linker comprises G and S residue.The length of connection peptides Spend extremely important to the activity of fusion protein.For the purpose of the present invention, it is preferable that the structure of described flexible peptide linker aminoacid composition Formula is (GS)a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c and d are greater than or equal to the integer of 0, and a+b+c+d >=1.
In some embodiments of the present invention, described peptide linker is selected from following sequence:
(a) L1:GSGGGSGGGGSGGGGS；
(b) L2:GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(c) L3:GSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(d) L4:GGGGSGGGGSGGGGSGGGGS.
Wherein, described human chorion gonadotrophic hormone beta-subunit carboxyl terminal peptide rigidity peptide (CTP) selects free human chorionic Full length sequence that gonadotrophin beta subunit carboxyl terminal the 113rd to 145 amino acids is formed or its fragment are (hereinafter referred to as CTP), specifically, described CTP rigidity peptide comprises SEQ ID NO:1 or the sequence of its truncate.
Preferably, described CTP rigidity peptide comprises at least 2 glycosylation sites；Such as, a preferred embodiment of the present invention In, described CTP comprises 2 glycosylation sites, and exemplarily, described CTP comprises 10 aminoacid of SEQ ID NO:1N end, i.e. SSSS*KAPPPS* (* represents glycosylation site)；Or described CTP comprises 14 aminoacid, i.e. S* of SEQ ID NO:1C end RLPGPS*DTPILPQ；And for example, in another embodiment, described CTP comprises 3 glycosylation sites, exemplarily, described CTP bag 16 aminoacid, i.e. SSSS*KAPPPS*LPSPS*R containing SEQ ID NO:1N end；For another example, in other embodiments, described CTP comprises 4 glycosylation sites, and exemplarily, described CTP sequence comprises 28,29,30,31,32 or 33 aminoacid and starts In the 113rd, 114,115,116,117 or 118 of human chorion gonadotrophic hormone beta subunit, terminate at the 145th.Specifically, Described CTP comprises 28 aminoacid, i.e. SSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ of SEQ ID NO:1N end.Every kind Probability all represents the standalone embodiment of the present invention.
In further embodiments, the CTP rigid element that the present invention provides is same with natural CTP aminoacid sequence at least 70% Source；In further embodiments, CTP rigid element and natural CTP aminoacid sequence at least 80% homology that the present invention provides；? In other embodiments, the CTP rigid element that the present invention provides and natural CTP aminoacid sequence at least 90% homology；At another In a little embodiments, the CTP rigid element that the present invention provides and natural CTP aminoacid sequence at least 95% homology.
Preferably, in embodiments of the invention, described CTP rigidity peptide preferably comprises following sequence units:
(i)CTP1: SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)CTP2: PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)CTP3: SSSSKAPPPS；
(iv)CTP4: SRLPGPSDTPILPQ.
Fusion protein of the present invention can also comprise the above-mentioned CTP rigidity peptide sequence unit of 2 or more than 2, such as this In one embodiment of invention, described CTP comprises 2 CTP3Unit: SSSSKAPPPSSSSSKAPPPS (CTP3-CTP3, or represent For (CTP3)2)。
Wherein, extend half-life part and preferably be selected from immunoglobulin IgG, IgM, IgA Fc fragment；More preferably from human IgG1, The Fc fragment of IgG2, IgG3 or IgG4 and variant thereof；Further, described human IgG Fc variant comprises and is positioned at wild type human IgG At least one in Fc is amino acid modified, and variant have reduction effector function (ADCC and/or CDC effect) and/or with The binding affinity of neonatal receptor FcRn strengthens.Further, human IgG Fc variant is selected from lower group:
(a)vFcγ1: containing Leu234Val, Leu235Ala and Pro331Ser sudden change human IgG1 hinge region, CH2 and CH3 region (aminoacid sequence as shown in SEQ ID NO:4)；
(b)vFcγ2-1: human IgG2 hinge region containing Pro331Ser sudden change, CH2 and CH3 region is (such as SEQ ID NO:5 Shown aminoacid sequence)；
(c)vFcγ2-2: containing Pro331Ser, Thr250Gln and Met428Leu sudden change human IgG2 hinge region, CH2 and CH3 region (aminoacid sequence as shown in SEQ ID NO:6)；
(d)vFcγ4: containing Ser228Pro and Leu235Ala sudden change human IgG 4 hinge region, CH2 and CH3 region (as Aminoacid sequence shown in SEQ ID NO:7).
More preferably, the aminoacid sequence of described hGH-L-CTP-vFc fusion protein is as shown in SEQ ID NO:2.
In a second aspect of the present invention, it is provided that recombinate described in a kind of coding first aspect present invention hGH-L-CTP-vFc The DNA molecular of fusion protein, it is preferable that described DNA sequence has the nucleotide sequence shown in SEQ ID NO:3.
According to the third aspect of the invention we, it is provided that a kind of carrier, this carrier comprises the DNA sequence described in second aspect present invention Row.
According to the fourth aspect of the invention, it is provided that a kind of host cell, this host cell comprises third aspect present invention institute State carrier, or transfect above-mentioned carrier.
In the detailed description of the invention of the present invention, host cell is the derived cell strain DXB-11 of CHO.
Preferably, described host cell in every 24 hours, produces thin more than 30 μ g/ million in its growth medium The restructuring hGH-L-CTP-vFc fusion protein as described in the first aspect of the invention of born of the same parents.
In a fifth aspect of the present invention, it is provided that one prepares restructuring hGH-L-CTP-vFc described in first aspect present invention The method of fusion protein, described method includes:
A the DNA of encoding fusion protein is introduced Chinese hamster ovary celI by (), generate CHO derived cell system；
In (b) screening step (a) in every 24 hours periods, express more than 30 μ g/106(million) high yield of individual cell is thin Born of the same parents' strain；
C cell strain that () incubation step (b) screens, expressed fusion protein；
D fermentation liquid that () results step (c) obtains, purified fusion protein.
In a sixth aspect of the present invention, the invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes pharmacy Upper acceptable carrier, excipient or diluent, and the described hGH-L-CTP-vFc fusion protein of effective dose.
Another further aspect, the invention provides described hGH-L-CTP-vFc fusion protein for treating because of endogenous growth The dysplasia that hormone secretion deficiency causes, and the build that Turner syndrome causes is short and small, chronic renal failure, Prader-Willi syndrome or the disease such as idiopathic build is short and small.
To sum up, the fusion part human IgG Fc that fusion protein structure of the present invention has the following characteristics that 1, fusion protein uses Variant is non-cracking performance, reduces the effector function being combined and trigger with Fc γ Rs and Clq；2, fusion prepared by the present invention No matter albumen is respectively provided with good stability during fermentation, purge process and storage；3, by L-CTP connect hGH with Fc variant, CTP rigidity peptide can not only extend its Half-life in vivo further；Obstruct by multiple glycosylation side chains simultaneously is made With, increase the space length between fusion molecule, promote each autofolding of hGH and Fc section to form correct three-dimensional conformation and be independent of each other Respective biological activity, compared with hGH-L-vFc, the biological activity of hGH-L-CTP-vFc increases substantially；4, fusion protein tool There is the immunogenicity of reduction so that it is the risk reduction that induction neutralizing antibody produces；5, compared with hGH-L-vFc, hGH-L-CTP- VFc has the bioavailability of raising, and blood concentration fluctuation is little, and safety improves, and improves toleration；There is the internal of prolongation follow The ring half-life, it is possible to decrease frequency of injection, improve patient compliance；6, compared with restructuring hGH, hGH-L-CTP-vFc purification step Simply, purification efficiency is high.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment) Can be combined with each other between each technical characteristic that body describes, thus constitute new or preferred technical scheme.As space is limited, exist This tires out the most one by one states.
Detailed Description Of The Invention:
IgG Fc variant
Non-cracking performance Fc variant
Fc element derives from the constant region fc fragment of immunoglobulin IgG, and it rises in the immune defence of eliminating pathogen Important function.The effector function of the IgG of Fc mediation plays by two kinds of mechanism: (1) and cell-surface Fc receptors (Fc γ Rs) In conjunction with, phagocytosis or splitting action or killing cell digest cause of disease by antibody dependent cellular cytotoxicity (ADCC) approach Body, or (2) be combined with the C1q of the first complement component C1, causes CDC (CDC) approach, thus cracks disease Substance.For the treatment being applied to people, the hGH being incorporated on target cell surface when hGH-L-CTP-vFc fusion protein is subject to During body, the Fc region of fusion protein does not the most have ill effect subfunction, from without dissolving or removing these target cell. Therefore, the Fc region of hGH-Fc must be non-deliquescent, to being incorporated into Fc γ Rs and Clq thus trigger effect subfunction side Face, Fc region is preferably inactive.In four kinds of human IgG hypotypes, IgG1 and IgG3 can effectively combine Fc γ Rs, IgG4 and Fc The binding affinity of γ Rs is relatively low, and the combination of IgG2 with Fc γ Rs is so low that to be difficult to measure, so human IgG2 is almost without ADCC Effect.Additionally, human IgG1 and IgG3 can also effectively combine C1q and activating complement cascade reaction.Human IgG2 combines relative with C1q Weak, and IgG4 is not combined (Jefferis R etc., Immunol Rev, 1998,163:59-76) with C1q, so human IgG2 CDC effect Should be the most weak.Obviously, a kind of natural IgG hypotype is not had to be especially suitable for producing hGH fusion protein.In order to not had effector merit The non-cracking performance Fc of energy, most effectual way is to complement, receptor binding domains sudden change transformation, regulation Fc and associated receptor in Fc fragment Binding affinity, reduce or eliminate ADCC and CDC effect, only retaining the long circulating half-life characteristics of Fc, and do not produce cell Toxicity.The comprised mutational site of more non-cracking performance Fc variant may refer to Shields RL etc., J Biol Chem, 2001, 276 (9): 6591-604 or Chinese invention patent CN 201280031137.2.
Pharmacokinetically improved Fc variant
The plasma half-time of IgG depends on the combination of it and FcRn, typically combines when pH 6.0, at pH 7.4 (blood plasma PH) dissociate time.By the research to both binding sites, the site that transformation IgG is combined with FcRn, it is allowed to when pH 6.0 tie Conjunction ability increases.The sudden change having turned out some residues for combining people's Fc γ domain important for FcRn can increase serum half Decline the phase.Especially, in people Fc γ 1, when three residues are substituted by other 19 conventional amino acid, some point mutation display increases FcRn binding affinity (Hinton etc., J Biol Chem, 279 (8): 6213-6216,2004).Hinton etc. find 2 mutants of T250Q and M428L make the combination with FcRn increase by 3 and 7 times respectively.2 sites of simultaneous mutation, then combine increase 28 times.In rhesus monkeys, M428L or T250QM428L mutant display plasma half-time increases by 2 times of (Paul R.Hinton Deng, J Immunol, 2006,176:346-356).Additionally, there is research that the Fc section of five kinds of humanized antibodies is carried out T250Q/ M428L sudden change not only improves the interaction of Fc Yu FcRn, and in internal pharmacokinetic trial subsequently, finds with skin Hemostasis is administered, and Fc mutant antibodies pharmacokinetic parameter compared with wild-type antibodies improves, and internal exposed amount increases, clearance rate Reduce, subcutaneous bioavailability be improved (.MAbs.Taylor&Francis such as Datta-Mannan A, 2012,4 (2): 267-273.)。
Carboxyl terminal peptide (CTP)
CTP is the small peptide of one section of β from human chorionic gonadotropin (hCG)-subunit carboxyl terminal.Four kinds and reproduction Relevant peptide hormone follicle stimulating hormone (FSH), lutropin (LH), thyrotropin (TSH) and chorionic gonadotrophin Hormone (hCG) contains identical α-subunit and the most special β-subunit.Compared with other three kinds of hormones, hCG Half-life in vivo Being obviously prolonged, this is mainly derived from distinctive carboxyl terminal peptide (CTP) (Fares FA etc., Proc Natl on its β-subunit Acad Sci USA,1992,89:4304-4308).CTP contains 37 amino acid residues, and it has 4 O-glycosylation sites, Terminal is sialic acid residues.Electronegative, the most sialylated CTP can resist the kidney scavenging action to it, thus extends The albumen half-life in vivo.Thus, inventor creatively increases at least one after the flexible peptide linker of suitable length CTP polypeptide so that the half-life of fusion protein extends further, bioavailability improves.
Additionally, by increasing CTP peptide between hGH and Fc variant, be equivalent to add one section of peptide that is rigidly connected.This is on the one hand Ensure that the hGH that N-end merges does not interferes with the binding site of Fc variant and FcRn, thus affect the half-life；Additionally Fc Protein A binding site is critically important for purification step in preparation technology, connects CTP and ensures that the hGH of N-end fusion also will not " cover " binding site of it and protein A.On the other hand, the interpolation of CTP also makes the Fc fragment of about 25KD size will not The correct folding of the hGH that interference N-end merges, so that the decline of its biologic activity/function or forfeiture.There is multiple glycosyl Side chain rigidity CTP polypeptide, relative to the random coil of this kind of flexible peptide linker of (GGGGS) n, it can form stable standing Body conformation, this " obstruct " effect promotes hGH and Fc section independently to fold the correct three-dimensional conformation of formation and be independent of each other respective Biological activity.
Fusion protein and production method thereof
Fusion protein of the present invention is generally prepared by biosynthetic method.According to nucleotide sequence of the present invention, this Technical field personnel can prepare the code nucleic acid of the present invention easily with various known methods.These methods such as but not limited to: PCR, DNA synthetic etc., concrete method can be found in J. Pehanorm Brooker, " Molecular Cloning: A Laboratory guide ".As the present invention A kind of embodiment, the method that can be carried out Overlap extension PCR by salvage nucleotide sequence again builds the present invention's Nucleic acid sequence encoding.
Present invention also offers a kind of expression vector, the sequence of the fusion protein comprising code book invention and operating therewith Property be connected expression regulation sequence.Described " being operatively connected " or " being operably coupled to " refers to such a situation, the most linearly Some part of DNA sequence can regulate or control the activity of same linear DNA molecule other parts.Such as, if promoter Control transcribing of sequence, then it is operably coupled to coded sequence exactly.
Expression vector can use commercially available such as but not limited to: pcDNA3, pIRES, pDR, it is thin that pUC18 etc. can be used for eucaryon The carrier of born of the same parents' system expression.Those skilled in the art can select suitable expression vector according to host cell.
According to the restriction enzyme mapping of known unloaded expression vector, those skilled in the art can be conventionally by restricted Enzyme is sheared and splicing, and the coded sequence of the fusion protein of the present invention is inserted suitable restriction site, prepares the weight of the present invention Group expression vector.
Present invention also offers the host cell expressing fusion protein of the present invention, wherein contain the fusion protein of the present invention Coded sequence.Described host cell preferably eukaryotic cell, such as but not limited to CHO, COS cell, 293 cells, RSF is thin Born of the same parents etc..As the optimal way of the present invention, described cell is Chinese hamster ovary celI, and it can express the fusion protein of the present invention well, Combination activity can be obtained good, the fusion protein having good stability.
The present invention also provides for a kind of method that recombinant DNA prepares fusion protein of the present invention, and its step includes:
1) nucleotide sequence (such as SEQ ID NO:3 sequence) of encoding fusion protein is provided；
2) by 1) nucleotide sequence be inserted into suitable expression vector, it is thus achieved that recombinant expression carrier；
3) by 2) recombinant expression carrier import suitable host cell；
4) transformed host cell is cultivated under conditions suitable for the expression；
5) supernatant is collected, and purified fusion protein product.
Described coded sequence importing host cell can be used the multiple known technology of this area, such as but not limited to: phosphorus Acid calcium deposit, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkali metal from Sub-method.
Cultivation and expression about host cell can be found in Olander RM Dev Biol Stand 1996；86:338. Clear liquid can be collected by the cell in centrifugal segregation suspension and residue.Can be identified by agarose gel electrophoresis technology.
Can be substantially uniform character by the above-mentioned fusion protein purification prepared, such as on SDS-PAGE electrophoresis in Single band.Such as, when recombiant protein is secreting, expressing, the ultrafilter membrane of commercialization can be used to separate described albumen, example Such as Products such as Millipore, Pellicon, first will express supernatant and concentrate.Concentrated solution can use the method for gel chromatography The most in addition purification, or use the method purification of ion-exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or sun from Sub-displacement chromatography.Gel-type vehicle can be the substrate that agarose, glucosan, polyamide etc. are usually used in protein purification.Q-or SP-group It it is ideal ion-exchange group.Finally, can also be used with hydroxylapatite adsorption chromatography, metal chelate chromatography, hydrophobic mutually The method polishing purification further to above-mentioned purified product such as effect chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC).Above-mentioned institute There is purification step to may utilize different combinations, finally make purity of protein reach substantially uniform.
The fusion to expressing of the available affinity column containing the specific antibody of described fusion protein, receptor or part Albumen is purified.According to the characteristic of the affinity column used, available conventional method, such as high-salt buffer, change pH etc. Method elution of bound amalgamation polypeptide on affinity column.Selectively, the aminoterminal of described fusion protein or c-terminus are also One or more polypeptide fragments can be contained, as protein tag.Any suitable label may be used to the present invention.Such as, institute The label stated can be FLAG, HA, HAl, c-Myc, 6-His or 8-His etc..These labels can be used for carrying out pure to fusion protein Change.
Pharmaceutical composition
Present invention also offers a kind of pharmaceutical composition, it contains effective dose (such as 0.000001-90wt%；Preferably 0.1-50wt%；More preferably, 5-40wt%) the fusion protein of the present invention, and pharmaceutically acceptable carrier.Generally, may be used The fusion protein of the present invention of effective dose is formulated in nontoxic, in inert and pharmaceutically acceptable aqueous carrier medium, its Middle pH ordinarily be about 5-8, it is preferred that pH is about 6-8.Term " effective dose " or " effective dose " refer to can be to people and/or animal Amount that is that produce function or activity and that can be accepted by people and/or animal.The composition of " pharmaceutically acceptable " applies to people And/or mammal and without excessive bad side reaction (such as toxicity, stimulation and allergy), i.e. there is rational benefit/wind The material of danger ratio.Term " pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and dilute Release agent.
Pharmaceutically acceptable carrier includes (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and A combination thereof.Generally pharmaceutical preparation should match with administering mode, and the pharmaceutical composition of the present invention can be to be made into injection form, example As being prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.Described drug regimen Thing the most aseptically manufactures.The dosage of active component is therapeutically effective amount.The pharmaceutical preparation of the present invention may also be fabricated which slow Release formulation.
The effective dose of fusion protein of the present invention can be with the pattern being administered and the order of severity etc. of disease to be treated And change.The preferably selection of effective dose can be determined according to various factors by those of ordinary skill in the art and (such as passes through Clinical trial).Described factor includes but not limited to: the pharmacokinetic parameter of described fusion protein such as biological utilisation Degree, metabolism, half-life etc.；The order of severity of the disease that patient is to be treated, the body weight of patient, the immune state of patient, administration Approach etc..Generally, when the present invention fusion protein every day with about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-10mg/kg the weight of animals) dosage give, gratifying effect can be obtained.Such as, compeling by treatment situation Highly necessary ask, the most separate dosage can be given every day, or dosage is reduced pari passu.
Accompanying drawing explanation
Fig. 1, show the fusion protein of Spe I-EcoR I fragment in pAPG-3 expression vector nucleotide sequence and The aminoacid sequence derived.Peptide linker position in maturation protein marks with lower stroke of dotted line.In Fc region, the nucleotide of runic Mark with corresponding amino acid variant underscore.
Fig. 2, show hGH fusion protein stimulate Nb2-11 cell proliferation ability.
Fig. 3, show the Drug-time curve of hGH fusion protein.
Fig. 4, show hGH fusion protein be administered after the growth curve of each group.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1, the expression plasmid of structure coding hGH fusion protein
Coding hGH leader peptide and the gene order of ripe hGH and different length flexible peptide linker, different length CTP are firm Property peptide and the gene order of different IgG Fc variants be all the Chinese hamster ovary celI preference codon of artificial optimization, through chemical synthesis process Obtain.For the ease of the specific site by purpose fragment inserting expressioning carrier, it is respectively arranged with a limit at synthesized fragment 5 ' and 3 ' end Property enzyme inscribe site processed, respectively SpeI and EcoRI.Fusion gene SpeI after sequence verification and EcoRI enzyme action, then insert Enter to PCDNA3.1 as template and between the corresponding restriction enzyme site of improved expression plasmid PXY1A1, obtain track fusion Plasmid pAPG.PXY1A1 plasmid is including but not limited to following important expression components and parts: 1) human cytomegalic inclusion disease virus early promoter and Enhancer needed for mammalian cell height heterogenous expression；2) Double Selection label, has kalamycin resistance in antibacterial, Mammalian cell has G418 resistance；3) mouse dihydrofolate reductase (DHFR) gene expression frame, when host cell is During DHFR gene defection type, methotrexate (MTX) can coamplification fusion gene and DHFR gene (see United States Patent (USP) US 4, 399,216)。
As shown in table 1, the present invention constructs a series of hGH-Fc fusion protein, and they have the flexible peptide of different length and connect Head, CTP composition and also position are different, and IgG Fc variant (vFc) the element composition of several different subtype.Wherein APG-3 The aminoacid sequence of nucleotide sequence and translation is as shown in Figure 1.
Table 1, several hGH fusion protein composition built
Embodiment 2, transient expression difference fusion protein and active determination in vitro
The a series of expression plasmids obtained in embodiment 1, use DNAFect LT reagent in the shaking flask of 30ml (ATGCell) transfection 3 × 107Chinese hamster ovary celI, the cell through transfecting grows 5 days in serum free growth medium, according to correspondence ELISA method measures the fusion protein concentration in supernatant, and measures the work of its extracorporeal biology by the method described in embodiment 5 Property.ELISA result shows that several plasmid transient expression amount under this condition is more or less the same, but their activity but demonstrates Bigger difference (table 2).Wherein, the molar specific activity of APG-1 is defined as 100% by us.The activity of APG-2 is APG-1's 80.9%, show only can not be effectively improved the activity of fusion protein by extending peptide linker, space steric effect the most each other is also Can not weaken along with the extension of peptide linker.Even, long peptide linker not only can not improve the activity of fusion protein, on the contrary can Make protein misfolding, with inactive multimeric forms secretion.It is presumed that reason, long flexible peptide linker makes fusion egg White flexibility ratio is higher so that it is can be freely rotatable, this may make the stereochemical structure of hGH closer to Fc district, and at the two it Between add CTP, on the one hand quite add one end rigidity peptide linker, make away from each other, it is often more important that CTP contains multiple glycosyl sides Chain, relative to the random coil form of flexible peptide linker, CTP can form fixing space conformation, it is possible to effectively separates The difference in functionality district of fusion protein, this is more beneficial for two parts and is independently folded into correct three-dimensional conformation, maintains higher work Property.The correctness of this supposition is demonstrated by the expression activitiy of APG-1, APG-2 and APG-3.And CTP is placed in Fc C end The activity of APG-4 only CTP is placed in the 61.3% of the APG-3 of Fc N end, and the activity of APG-4 is similar to the APG-1 without CTP. And specific activity APG-4 of APG-3 is high, the activity of APG-5, APG-6 and APG-7 to be also far above other fusion protein.More than tie Really, it was demonstrated that CTP is the most crucial to the activity of fusion protein, and CTP is placed in the N end of Fc and is more beneficial for improving the work of fusion protein Property.
Table 2, the external activity EC of various fusion protein of transient expression50Value compares
The expression in transfectional cell series of embodiment 3, fusion protein
The expression plasmid of restructuring is transfected into mammalian host cell line, to express hGH-L-CTP-vFc fusion protein. In order to stablize high-caliber expression, preferred host cell system be DHFR deficient CHO-cell (see United States Patent (USP) US 4, 818,679).A kind of preferably transfection method is electroporation, it is possible to use other method, turns including calcium phosphate cosedimentation, fat Dye and Protoplast fusion.In electroporation, with being set to 300V electric field and the Gene Pulser of 1050 μ Fd electric capacity Electroporator (Bio-Rad Laboratories, Hercules, CA), places 5 × 10 in cuvette7Individual cell, It is subsequently added the 50 μ linearizing plasmids of g BspCI and carries out electroporation.In transfection two days later, culture medium is made into containing 0.6mg/mL The growth medium of G418.After transfecting about 2 weeks, the transfectant to G418 medicine with resistance can grow up to macroscopic cell mass Fall.By the elisa assay method of anti-human igg Fc, screen the transfectant selecting medication to have resistance.Also can be with anti-hGH's ELISA carries out the quantitative analysis of fusion protein expression.By Method of Limited Dilution 96 well culture plate, sub-clone produces high level Fc and melts The hole of hop protein.
In order to realize the expression of fusion protein higher level, preferably carry out coamplification with the DHFR gene by MTX Drug inhibition. In the growth medium containing progressive concentration MTX, with the antigen-4 fusion protein gene of DHFR gene coamplification transfection.Use Method of Limited Dilution The transfectant that can grow in up to 6 μMs of MTX culture medium of sub-clone.Measure secretion rate the cell line of sub-clone is entered The analysis of one step.Secretion rate horizontal exceeding about 30 (being preferably about 50) μ g/106The cell line of (i.e. million) individual cell/24 hour Adapt to use the suspension culture of serum free growth medium.Use conditioned medium purified fusion protein the most again.
Embodiment 4, fusion protein purification with qualitative
With 1N NaOH, the conditioned medium containing fusion protein is titrated to pH 7～8, then with the nitric acid of 0.45 micron Cellulose filter filters.Filtrate is loaded onto on the Protein A post that phosphate buffer saline (PBS) balances.To be fused Protein binding, after Protein A post, discards the component of outflow.This post is washed, until the OD value at 280nm is less than with PBS 0.01.Then with the fusion protein of the citrate buffer solution elution of bound that 0.1M pH is 3.75.The eluent 1M of 0.4 volume K2HPO4Neutralize, merge the component containing purifying protein, and use PBS.Then with the nitrocellulose filter of 0.22 micron Filter, and be stored in 4 DEG C.Under non reducing conditions, SDS-PAGE protein product identified and analyze.Use BSA conduct Standard, by BCA protein analysis quantitatively this fusion protein.
Embodiment 5, the Bioactivity analysis of fusion protein
Nb2-11 cell proliferation is utilized to detect the In vitro biological activity of restructuring hGH-L-CTP-Fc fusion protein.
Mouse lymphoma cell Nb2-11 (the U.S. is normally cultivated by the Fischer's culture medium containing 10% hyclone ATCC cell bank).Using serum-free medium, 1000ng/ml initiates 3 times of gradient dilution fusion protein, it is thus achieved that 8 variable concentrations Sample, every hole 100 μ l, add to 96 orifice plates, culture medium is negative control.Take the cell being in logarithmic (log) phase trophophase, by nothing Once, adjust density is every milliliter 3 × 10 to blood serum medium washed cell6Individual cell, every hole 100 μ l joins above-mentioned 96 orifice plates In.At 37 DEG C, 5%CO2Cultivating 48 hours in incubator, (Cell Counting Kit, purchased from Shanghai to use CCK-8 test kit Yi Sheng bio tech ltd) detection cell proliferative conditions.The absorbance at 450nm is measured, by OD reading by microplate reader Mapping relative to the concentration of fusion protein, gained dose-effect curve calculates the biological activity of fusion protein.
Fig. 2 shows that hGH fusion protein stimulates the ability of Nb2 cell proliferation.Table 3 is that the half of different fusion protein is effective Concentration value (half maximal effective concentration, EC50).Due to the aminoacid of growth hormone C end and its Function is closely related, and Fc is directly connected with the C section of hGH can affect its biological activity.After adding connection peptides in hGH Yu Fc, hGH The activity raising of fusion protein.It can be seen from the results that the activity of APG-3 is compared with APG-1, activity improves will by about one time.This Being likely due to CTP mono-aspect and play the function of self, on the other hand as the peptide that is rigidly connected between Fc and hGH, CTP is with soft Property peptide coupling, this structure makes fusion protein be folded into more favourable three dimensional structure, it is ensured that the biological activity of hGH.
Table 3, the EC of hGH fusion protein50Value
Embodiment 6, the pharmacokinetics of fusion protein measure
Male SPF level SD rat (purchased from Shanghai Bi Kai laboratory animal company limited), after raising in advance one week, often group selects body weight About 294g rat 3, single 0.176mg/kg gives intravenous injection APG-1 and APG-3 fusion protein, investigates blood medicine dense Degree and the Changing Pattern of time.Matched group and administration group the most upon administration 0,0.5,1,2,3,4,5,8,10,24,48,72h warp Eye socket is taken a blood sample, and blood 5000r/min after room temperature places 30min is centrifuged 10min, isolates serum ,-20 DEG C of preservations.With for ELISA method special for hGH measures hGH content in each time point serum.By software PKSOLVER, calculate each group of main medicine generation Kinetic parameter.Each group pharmacokinetic parameter result such as table 4.
Table 4, the pharmacokinetic parameter of hGH fusion protein
Being found out by result, the Half-life in vivo of APG-3 and APG-1 fusion protein is respectively 2.6 and 2.4 hours, the half-life Basically identical, this point can also be found out from MRT parameter, the two in vivo residence time also close to.When the medicine of Fig. 3 Find on curve that the blood drug level of APG-3 is always high than APG-1, thus it is speculated that the change being probably APG-3 structure result in its medicine generation Dynamic (dynamical) character there occurs change；The CLTB of APG-3 only has the half of APG-1, shows the internal removing phase of APG-3 To slowly, and the apparent volume of distribution of APG-3 is the twice of APG-1, illustrate APG-1 enter internal after be distributed to tissue rapidly In, result in its blood drug level less than APG-3.Apparent volume of distribution and CLTB there occurs change, and the two acts on jointly Result be that the blood drug level of APG-3 is higher than APG-1, the internal exposed amount (AUC) of medicine is the highest simultaneously.
APG-1 is after the sudden change in Fc site, thus it is speculated that it compares exposed amount increase, clearance rate reduction than unmutated. APG-3 adds the structure of CTP on the basis of APG-1, reduces clearance rate further and increases AUC, reaching above-mentioned two After individual purpose, the most further reduce apparent volume of distribution owing to introducing CTP rigid structure so that it is be distributed more in the tissue Few, medical content in blood is higher, causes internal exposed amount higher, so the structural grouping advantage of APG-3 is not only embodied in excellent Medicine more is in parameter, and its drug effect may be also superior to APG-1, because APG-3 blood drug level after transformation is higher, and meaning Free medicine in vivo more than APG-1, so the drug effect of APG-3 is better than APG-1, bioavailability is higher, it is contemplated that Its clinical administration dosage will also decrease.As can be seen here, APG-3 shows excellence in terms of biologic activity and pharmacokinetics Performance.
Embodiment 7, the Biological acdtivity in vivo of fusion protein measure
Choosing 4 week old SPF level male SD rats, body weight 60-80g, by National Institute for Food and Drugs Control's laboratory animal Center provides.
Testing first 2 weeks clean conditions menisectomy Hypophysectomies, removing hypophysis Post operation 2 weeks is convalescent period, selects big before being administered Mus body weight change, less than the qualified healthy animal of operation consent body weight ± 10%, goes hypophysis rat to be divided into 6 groups by body weight is uniformly random, 5/group.The positive reference tested as this with fugitive rhGH (trade name norditropin, Novo Nordisk A/S produces) Medicine 1 (Y1), than 3IU/mg alive；Using PEG-rhGH (Changchun Jinsai Medicine Co., Ltd) as positive with reference to medicine 2 (Y2), than Live 6IU/mg；APG-3 is basic, normal, high, and dosage setting is respectively 5mg/kg/14d, 15mg/kg/14d and 45mg/kg/14d.According to Molecular size range and molal quantity, the middle dosage 15mg/kg/14d of APG-3 is suitable with reference to medicine Y1 and Y2 with positive.Administering mode is Cervical region subcutaneous injection, various dose APG-3 fusion protein and Y2, every Mus was administered once in the 1st day and the 8th day；Y1 is administered every day 1 time, continuous 14 days.
After administration, every rat is weighed every day, the 15th day with carbon dioxide suffocate execution whole rats, weigh.Every animal I-th day (di) body weight (bwi) i.e. 1st day (d1) body weight (bw front with administration1) the body weight that i.e. increases of difference (if desired, experiment terminates After can perform an autopsy on sb., cut sella region, macroscopy with or without hypophysis remain, reject have hypophysis remaining animal).Body weight increases meter Calculation formula: Δ bw=bwi–bw1, wherein: Δ bw is weight gain；bw1For d1 body weight before being administered；bwiFor being administered latter i-th day body Weight.Measurement data is with mean standard deviationRepresent, the results are shown in Table 5.Fig. 4 is the growth curve of each group after showing administration.
Rat body weight change before and after the administration of table 5, hGH fusion protein (N=7)
From the results, it was seen that compared with model group, the 8th day each administration group Δ bw all dramatically increases upon administration, but respectively Between administration group, Δ bw value difference is different the most notable；And the 15th day upon administration, APG-3 has highly significant to the body weight growth of rat Facilitation, it is rhGH (Y1) and PEG-rhGH (Y2) that high dose APG-3 induction Hypophysectomized rats body weight increases (Δ bw value) About 1.5 times of group；And the body weight increase of middle dosage APG-3 induction is slightly better than Y1, basically identical with Y2.It was found that APG-3 is high Rat body weight is increased after being administered at the 2nd time by dosage group, shows more significant facilitation compared with PEG-rhGH group, and Being administered in one week after for 1st time, the Δ bw value of two groups does not the most have significant difference.Deduce from this diversity interpretation of result APG-3 compares PEG-rhGH should have the longer activity in vivo half-life, and therefore after repeat administration, this body accumulation effect makes Obtain the ascendant trend of growth curve after APG-3 group is administered at the 2nd time more precipitous.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally Enclose.

Claims (13)

1. recombinant human somatropin's fusion protein, it is characterised in that described fusion protein contains people from N end successively to C end Growth hormone, flexible peptide linker, the carboxyl terminal rigidity peptide of at least one human chorion gonadotrophic hormone beta subunit and people's immunity ball Albumen Fc fragment；Wherein Fc fragment preferred human IgG Fc variant；It is highly preferred that described human IgG Fc variant comprises is positioned at wild type At least one in human IgG Fc is amino acid modified, and variant has effector function and/or and the neonatal receptor of reduction The binding affinity of FcRn strengthens.
2. fusion protein as claimed in claim 1, it is characterised in that described flexible peptide linker contains two or more selected from sweet The aminoacid of propylhomoserin, serine, alanine and threonine, it is preferred that the general structure of flexible peptide linker aminoacid composition is (GS) a (GGS) b (GGGS) c (GGGGS) d, wherein a, b, c and d are greater than or equal to the integer of 0, and a+b+c+d >=1,
It is highly preferred that the aminoacid of described flexible peptide linker is selected from following sequence:
(a)GSGGGSGGGGSGGGGS；
(b)GSGGGGSGGGGSGGGGSGGGGSGGGGS；
(c)GSGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS；
(d)GGGGSGGGGSGGGGSGGGGS。
3. fusion protein as claimed in claim 1, it is characterised in that the carboxyl of described human chorion gonadotrophic hormone beta subunit End rigidity peptide comprises SEQ ID NO:1 or the sequence of its truncate, and the sequence of wherein said truncate comprises at least 2 glycosylation positions Point,
Preferably, the carboxyl terminal rigidity peptide of described human chorion gonadotrophic hormone beta subunit is selected from following sequence units:
(i)SSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(ii)PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ；
(iii)SSSSKAPPPS；
(iv)SRLPGPSDTPILPQ。
4. fusion protein as claimed in claim 1, it is characterised in that the carboxyl of described human chorion gonadotrophic hormone beta subunit Terminal peptide rigid element at least has 70%, 80%, 90% with the CTP aminoacid sequence in fusion protein described in claim 3 Or the homogeneity of 95%.
5. fusion protein as claimed in claim 1, it is characterised in that described fusion protein comprises 1,2,3,4 or 5 human chorionics The carboxyl terminal peptide rigid element of film gonadotrophin beta subunit.
6. fusion protein as claimed in claim 1, it is characterised in that described human IgG Fc is selected from lower group:
A () contains the human IgG1 hinge region of Leu234Val, Leu235Ala and Pro331Ser sudden change, CH2 and CH3 region；
B () contains the human IgG2 hinge region of Pro331Ser sudden change, CH2 and CH3 region；
C () contains the human IgG2 hinge region of Pro331Ser, Thr250Gln and Met428Leu sudden change, CH2 and CH3 region；
D () contains human IgG 4 hinge region of Ser228Pro and Leu235Ala sudden change, CH2 and CH3 region.
7. fusion protein as claimed in claim 1, it is characterised in that the aminoacid sequence of described fusion protein such as SEQ ID Shown in NO:2.
8. the DNA molecular of the fusion protein encoded described in any one of claim 1-7, it is preferred that described DNA molecular tool There is the nucleotide sequence shown in SEQ ID NO:3.
9. a carrier, it is characterised in that comprise DNA molecular as claimed in claim 8.
10. a host cell, it is characterised in that comprise carrier as claimed in claim 9, or transfected claim 9 Described carrier.
Prepare the method for recombination fusion protein as described in claim 1-7 for 11. 1 kinds, it is characterised in that comprise the steps:
A the DNA of encoding fusion protein is introduced Chinese hamster ovary celI by (), generate CHO derived cell system；
In (b) screening step (a) in every 24 hours periods, express the high yield cell more than 30 μ g/106 (million) individual cells Strain；
C cell strain that () incubation step (b) screens, expressed fusion protein；
D fermentation liquid that () results step (c) obtains, purified fusion protein；
Preferably, the CHO derived cell system in step (a) is DXB-11.
12. 1 kinds of pharmaceutical compositions, it is characterised in that described compositions contains with good grounds claim 1-7 any one claim Described fusion protein and pharmaceutically acceptable carrier.
13. fusion protein as described in claim 1-7 are for preparing the medicine for the treatment of endogenous growth hormone hyposecretion Purposes.
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